Toward the prediction of the activity of antioxidants: experimental and theoretical study of the gas-phase acidities of flavonoids.

The relative gas-phase acidities were determined for eight flavonoids, applying the kinetic method, by means of electrospray-ion trap mass spectrometry. The experimental acidity order, myricetin > luteolin > quercetin > (+/-)-taxifolin > kaempferol > apigenin > (+)-catechin > (+/-)-naringenin shows good agreement with the order obtained by theoretical calculations at the B3LYP/6-311 + G(2d,2p)//HF/6-31G(d) level. Moreover, these calculations provide the gas-phase acidities of the different OH groups for each flavonoid. The calculated acidity values (Delta(ac)H), corresponding to the most favorable deprotonation, cover a narrow range, 314.8-330.1 kcal/mol, but the experimental method is sensitive enough to differentiate the acidity of the various flavonoids. For all the flavones and the flavanol, catechin, the 4'-hydroxyl group is the most favored deprotonation site whereas for the flavanones studied, taxifolin and naringenin, the most acidic site is the 7-hydroxyl group. On the other hand, the 5-hydroxyl, in flavones and naringenin, and the 3-hydroxyl, in taxifolin and catechin, are always the less acidic positions. The acidity pattern observed for this family of compounds mainly depends on the following structural features: The ortho-catechol group, the 2,3 double bond and the 4-keto group.

Diffractive micro bar codes for encoding of biomolecules in multiplexed assays.

Microparticles incorporating micrometer-sized diffractive bar codes have been modified with oligonucleotides and immunoglobulin Gs to enable DNA hybridization and immunoassays. The bar codes are manufactured using photolithography of a chemically functional commercial epoxy photoresist (SU-8). When attached by suitable linkers, immobilized probe molecules exhibit high affinity for analytes and fast reaction kinetics, allowing detection of single nucleotide differences in DNA sequences and multiplexed immunoassays in <45 min. Analysis of raw data from assays carried out on the diffractive microparticles indicates that the reproducibility and sensitivity approach those of commercial encoding platforms. Micrometer-sized particles, imprinted with several superimposed diffraction gratings, can encode many million unique codes. The high encoding capacity of this technology along with the applicability of the manufactured bar codes to multiplexed assays will allow accurate measurement of a wide variety of molecular interactions, leading to new opportunities in diverse areas of biotechnology such as genomics, proteomics, high-throughput screening, and medical diagnostics.

Covalent attachment of proteins to solid supports and surfaces via Sortase-mediated ligation.

BACKGROUND: There is growing interest in the attachment of proteins to solid supports for the development of supported catalysts, affinity matrices, and micro devices as well as for the development of planar and bead based protein arrays for multiplexed assays of protein concentration, interactions, and activity. A critical requirement for these applications is the generation of a stable linkage between the solid support and the immobilized, but still functional, protein. METHODOLOGY: Solid supports including crosslinked polymer beads, beaded agarose, and planar glass surfaces, were modified to present an oligoglycine motif to solution. A range of proteins were ligated to the various surfaces using the Sortase A enzyme of S. aureus. Reactions were carried out in aqueous buffer conditions at room temperature for times between one and twelve hours. CONCLUSIONS: The Sortase A transpeptidase of S. aureus provides a general, robust, and gentle approach to the selective covalent immobilization of proteins on three very different solid supports. The proteins remain functional and accessible to solution. Sortase mediated ligation is therefore a straightforward methodology for the preparation of solid supported enzymes and bead based assays, as well as the modification of planar surfaces for microanalytical devices and protein arrays.

Multistep synthesis on SU-8: combining microfabrication and solid-phase chemistry on a single material.

SU-8 is an epoxy-novolac resin and a well-established negative photoresist for microfabrication and microengineering. The photopolymerized resist is an extremely highly crosslinked polymer showing outstanding chemical and physical robustness with residual surface epoxy groups amenable for chemical functionalization. In this paper we describe, for the first time, the preparation and surface modification of SU-8 particles shaped as microbars, the attachment of appropriate linkers, and the successful application of these particles to multistep solid-phase synthesis leading to oligonucleotides and peptides attached in an unambiguous manner to the support surface.